Plant genetic engineering has provided great potential for the improvement of commercially important plant species. In recent years, the genetic engineering of trees has gained momentum and finds particular application in the pulping and timber industries. Several established tree transformation systems exist for such species as sweetgum (Sullivan and Lagrinini 1993), European larch (Huang et al. 1991), yellow poplar (Wilde et al. 1992) and many Populus sp. (Minocha et al. 1986, Fillatti et al. 1988, De Block 1990, Brasileiro et al. 1992, Tsai et al. 1994). Species in the Populus genera have served as the model systems for the genetic engineering of trees (Kim et al. 1997). Various traits, such as insect resistance and herbicide tolerance, have been engineered into these tree species (Klopfenstein et al. 1993, De Block 1990). Thus, the potential for genetically engineering tree species is great for commercially important tree species, including Eucalyptus. 
Eucalyptus plants are polygenus plants comprising more than five hundred species. Eucalyptus has a high growth rate, adapts to wide range of environments, and displays little susceptibility to insect damage. In addition to its exceptional growth properties, Eucalyptus trees provide the largest source of fibers for the paper industry. Fibers from hardwood species, such as Eucalyptus, are generally much shorter than fibers from softwoods, such as pine. The shorter fibers produced from Eucalyptus results in the production of pulp and paper with desirable surface characteristics, including smoothness and brightness, but low tear or tensile strength. As a timber, Eucalyptus provides tall, straight timber with a medium to high density. Eucalyptus timber is general-purpose; finds use in the plywood and particleboard industry, furniture industry; and provides a source of firewood and construction materials.
Most reports demonstrating transformation of Eucalyptus use Eucalyptus seedlings rather than elite genotypes obtained through breeding programs. For example, WO 99/48355 describes a method for transforming young leaf explants from seedlings of E. grandis and E. camaldulensis. Even though the transformation was successful, there are two main problems with the described method. First, the regeneration protocol works for seedling explants, but it does not work with explants from elite genotypes. Second, even with seedling explants and the claimed improvements, the transformation efficiency is limited to 2.2% or lower for cotyledon explants of the two species and the hypocotyl explants of E. camaldulensis. An improved transformation and regeneration protocol was reported for E. camaldulensis seedlings by Ho et al., Plant Cell Reports 17:675-680 (1998); but, the protocol was not repeatable even with E. camaldulensis seedlings. Thousands of explants were cultured, and transgenic callus lines were produced, but the number of shoots recovered was minimal. Hartcourt et al. Molecular Breeding 6:306-315 (2000) reported the transformation of E. camaldulensis seedlings with the insecticidal cry3A gene and the herbicide resistant bar gene. Although five herbicide resistant callus lines were regenerated from an unnumbered explants derived from fifty seedlings, one line was difficult to propagate in culture or in the greenhouse, and another line was proven to be an escape. Moreover, the line that was difficult to propagate was one of the two lines that were analyzed at molecular level, and it was the only line with a single insertion. These studies indicate that even when the recovery of transgenic Eucalyptus plants is possible, the low transformation efficiency precludes delivery of a desired gene into many genotypes. Thus, a need continues to exist for a genotype-independent method of Eucalyptus transformation and regeneration.
Although micropropagation of Eucalyptus seedlings has been performed, de novo shoot regeneration has been limited to seedlings instead of the selected or “elite” clones of commercially important Eucalyptus species. Elite genotypes, which arise through successive rounds of breeding, are valued for their combination of economically desirable traits. Unlike seedling transformation, which requires a large number of genotypes to ensure co-segregation of growth traits with the desired trait conferred by transgene expression, the transformation of elite clones would provide an efficient and advantageous system for genetically engineering tree species. Elite genotypes can be selected based on many years of clonal field test with a large number of starting genotypes. Like many other fast growing hardwood tree species, it takes years of field evaluation before relatively accurate predictions of a trait can be made for Eucalyptus. Therefore, if seedlings are used for genetic engineering, an even larger number of genotypes is needed for successful selection of a growth trait together with a desired trait conferred by transgene expression.
Both GB2298205 (WO/9625504) and EP 1050209 claimed the use of 1-(2-chlorl-4-pyridyl)-3-phehylurea or N-(2-chloro-4-pyridyl)-N′-phenylurea (4-PU or 4CPPU) as the primary cytokinin for regeneration of transgenic shoots. In both cases, antibiotics were used as selection agents. WO/9625504 demonstrates the transformation of explants from mature genotypes, but the inventors used seedling explants for E. globules and E. nitens to demonstrate transformation. EP 1050209 uses a vertical rotary culture system for inducing formation of transgenic primordia. Although transformation was demonstrated with rejuvenated explants from mature trees, the transformation efficiency was calculated based on transgenic callus production and there was no indication of the frequency for transgenic plant production. Since efficient de novo shoot regeneration is critical for genetic engineering, there is a need to develop a highly efficient regeneration system for the selection of clones from commercially important Eucalyptus species.
In addition to the need for an improved transformation system, there is a need for improved methods for selecting transformed plants. Most plant transformation protocols use antibiotic selection, incorporating an antibiotic into the selection media and an antibiotic resistance gene into the transformation gene construct. A common selection method uses nptII or hptII as a selectable marker and kanamycin or geneticin, or hygromycin, respectively, as a selection agent. While antibiotic selection provides a means for selecting transformed cells, it has several limitations. First, the incorporation of an antibiotic resistance gene in a transgenic organism is disfavored by the general public due to widespread concern about antibiotics and antibiotic resistance genes spreading from the transgenic organism into the environment. Second, antibiotic selectable markers provide no commercially desirable phenotype to the transformed plant, as they function only in the selection of transformed cells. Indeed, constitutive production of the antibiotic resistance protein may result in detriment to the value of the transformed plant in that it may divert a significant biomass from the commercially desirable phenotype.
To address these limitations, practitioners have investigated additional selectable markers for use in the production of transgenic plants. A popular replacement for the antibiotic selectable marker is herbicide resistance, such as that mediated by certain mutant genes encoding such enzymes as acetolactate synthase (ALS). The ALS enzyme catalyzes the first common step in a plant's biosynthetic pathway for producing the branched-chain amino acids valine, leucine and isoleucine. A number of effective and widely used herbicides target the ALS enzyme, including sulfonylureas, imidazolinones, and triazolopyrimidines.
Another type of resistance gene that has been explored for use in selection of transgenic plants shows resistance to a metabolic inhibitor that mimics natural feedback inhibition during production of a biosynthetic product, via a mutant gene that overcomes the metabolic inhibitor via constitutive overproduction of the product. An example of such a gene is anthranilate synthase (ASA), which mediates a critical step in the production of tryptophan, and is normally subject to feedback inhibition by tryptophan. Besides acting as selectable markers, such genes can confer a desirable growth phenotype on the transgenic plant it if the biochemical product, in this case tryptophan, is normally a limiting factor to the plant's growth.
While non-antibiotic selectable marker genes overcome some of the problems associated with using antibiotic resistance markers, the use of non-antibiotic selectable marker genes has not been a panacea for plant transformations. One problem associated with these markers is a high rate of false positives. Thus, practitioners are forced to evaluate many transformants to identify a true positive. Such excessive screening greatly increases the time and cost associated with creating transgenic plants.
Accordingly, there is a need for improved methods of selecting transformed plants.
Accordingly, there is a need to increase the frequency of transforming Eucalyptus cells and regenerating stably transformed plants from clones of elite germplasm.